The long-term goal of this project is to define molecular mechanisms that control synapse development and homeostasis. Drosophila NMJ is a glutamatergic synapse, similar in structure and physiology to mammalian central excitatory synapses. In flies each NMJ is unique and identifiable, synapses are large and accessible for electrophysiological and optical analysis, making the Drosophila NMJ a powerful genetic system to study synapse development. The Drosophila NMJ can thus be used to analyze and model defects in the structural and physiological plasticity of glutamatergic synapses, which are associated with a variety of human pathologies from learning, memory deficits to autism. The similarity in gross architecture, function, and molecular machinery supports the notion that studying the assembly and development of fly glutamatergic synapses will shed light on their vertebrate counterparts. In flies as in humans, synapse strength and plasticity is determined by the interplay between different iGluRs subtypes. The subunits that form the Drosophila glutamate-gated ion channels (iGluRs) are known, but only the NMJ receptor complexes have been well studied in vivo. They NMJ receptors consist of four different subunits: GluRIIC, -IID and -IIE, and either GluRIIA (in type-A receptors) or IIB (type-B). Various mechanisms regulating the extent of type-A and type-B receptors accumulation at synaptic sites have been described but the molecular mechanisms for the initial localization and clustering of receptors at synaptic sites remained unclear. We have recently discovered an obligatory auxiliary protein, Neto (Neuropillin and Tolloid-like), absolutely required for the iGluRs clustering and NMJ functionality. Neto belongs to a family of highly conserved proteins sharing an ancestral role in formation and modulation of glutamatergic synapses. Our investigations uncovered essential roles for Neto during synapse development and strongly support the notion that trafficking of both iGluR subtypes on the muscle membrane, their synaptic recruitment and stabilization, and their function are tightly regulated by Neto. Our results further suggest that the fly Neto isoforms (alpha and beta) directly engage iGluRs as well as other intracellular and extracellular proteins to selectively regulate the distribution of iGluRs subtypes, the recruitment of postsynaptic proteins, and the organization of postsynaptic structures. Since iGluRs gating properties control the distribution and trafficking of these receptors in vivo, Neto could influence the synaptic recruitment of iGluRs by simultaneously controlling multiple steps in receptor trafficking and clustering and/or receptor function. Until recently, our investigations were limited by the inability to reconstitute functional Drosophila NMJ receptors in heterologous systems and identify the structural elements and the auxiliary subunits important for receptor assembly, surface delivery, synaptic recruitment and function. We have recently solved this problem by accomplishing the first functional reconstitution of NMJ iGluRs in Xenopus oocytes. Furthermore, we have succeeded in expressing functional receptors in HEK293 cells, where the biophysical properties of these receptors can be further dissected using fast glutamate applications. The ability to examine the functional characteristics of iGluRs in heterologous systems opens up tremendous opportunities to study the modulation of iGluRs function and parse out a role for Neto and/or other auxiliary proteins in the receptor function vs. receptor assembly, surface expression, synaptic trafficking and/or stabilization. In addition, we searched for novel proteins important for NMJ development using a synthetic lethality screen that takes advantage of the 50% lethality of an allele with suboptimal levels of Neto. Using this strategy we have previously uncovered genetic interactions between neto and several BMP pathway components. Recently, we have tested a number of extracellular matrix components and discovered an interaction between neto and tenectin (tnc), which encodes a Mucin-type protein with RGD motifs. Our data indicate that Tnc is secreted from both motor neurons and muscles and accumulates at synaptic terminals. Tnc selectively recruits PS2/PS integrin at synaptic terminals, but only the cis Tnc/integrin complexes appear to be biologically active. These complexes have distinct pre- and postsynaptic functions, mediated at least in part through the local engagement of the spectrin-based membrane skeleton: the presynaptic complexes control neurotransmitter release, while postsynaptic complexes ensure the size and architectural integrity of synaptic boutons. This study reveals an unprecedented role for integrin in the synaptic recruitment of spectrin-based membrane skeleton.